The long term goals are to determine the molecular structure of the outer arm dynein of cilia and flagella, and to elucidate the functions of the individual subunits, polypeptides, and domains of polypeptides in this important mechanochemical transducer. The work will concentrate on the outer arm dynein of Chlamydomonas, which structurally and compositionally is the best characterized of all dyneins. One of the dynein intermediate chains, IC 78, is located at the base of the arm and is the major polypeptide cross-linked to tubulin in the axoneme. A full-length cDNA clone encoding IC 78 has been isolated and sequenced; it will now be determined if there is an existing null mutant for IC 78. If not, one will be produced by homologous recombination or antisense-RNA technology. The cross-linked IC 78/tubulin peptides will be isolated and sequenced, and site-directed mutagenesis and transformation of the null mutant used to determine the significance of the IC 78/tubulin interaction. The IC 78 gene also will be randomly mutagenized and introduced into the null mutant to determine what roles other parts of IC 78 have in arm assembly and function. Existing monoclonal antibodies and immunoelectron microscopy will be used to locate regions of the dynein heavy chains in the isolated particles. An existing genomic clone encoding part of the gamma heavy chain will be used to isolate cDNA clones covering the entire gamma chain transcript; these will be sequenced and analyzed to sites of the gamma chain will be isolated and sequenced. Site-directed mutagenesis and transformation will be used to eliminate the gamma chain's ATP-hydrolytic activity and thus determine the role of that chain in outer arm function. Cells also will be transformed with gamma chain genes in which judiciously chosen regions have been deleted or randomly mutagenized, and selected transformants analyzed to identify other functionally important domains. The force-producing and microtubule-binding properties of individual dynein subunits will be examined under different physiological conditions to better understand the roles of each subunit in the generation and control of flagellar movement. Dynein arms generate the forces that are the basis for motility in all eukaryotic cilia and flagella. Because the basic structure and composition of dynein appears to have been conserved throughout evolution, the fundamental information obtained from these studies will be applicable to the dyneins of other organisms, including man. Consequently, knowledge obtained from these studies will increase our understanding of human diseases such as immotile cilia syndrome, in which the arms are frequently lacking. The studies will also provide a basis for understanding such important processes as sperm maturation and capacitation, which are necessary for fertilization and which ultimately must involve changes in the functioning of the arms.